Methods for performing differential capture proteomics

Abstract

Disclosed herein are improved methods for identifying, isolating, and comparing proteins and other biomolecules differing between two biological samples using affinity chromatography and phage display techniques.

Description

BACKGROUND OF THE INVENTION [0001] The invention relates to methods for identifying and isolating proteins and other biological molecules using affinity chromatography and phage display techniques. [0002] There is an increasing need, particularly in the fields of medicine and agriculture, to identify and characterize the molecules which participate in a wide variety of biological processes and to find new molecules capable of modulating these processes. One way to search for novel bioactive compounds is to screen libraries of natural materials or synthesized molecules, using assay techniques which can range in complexity from simple binding reactions to elaborate physiological preparations. Unfortunately, selecting molecules of interest from large ensembles or repertoires can be time-consuming and costly, and often only provides leads which require further investigation and development. [0003] Recently, there have been several developments both in the generation of libraries and in ...

AI Technical Summary

Benefits of technology

[0021] The present invention provides various improvements to the DCP methodology. In particular, the present invention features methods for increasing the efficiency of the detection of biomolecules, e.g., proteins. For example, the DCP process may include the step of derivatization of protein species present in a biological sample, thereby modifying functional groups on the biomolecules in order to facilitate immobilization onto a support, followed by exposing the immobilized biomolecules from the sample to a library, such as a phage display library. This derivatization step is controlled by varying, for example, the derivatizing agent, the concentrations of reactants, the temperature, and time for derivatization. The matrices of the invention can be prepared by covalently linking biomolecules in a biological sample to a support. The samples are preferably treated with chemical agents to denature the biomolecules, e.g., proteins, prior to being adhered to the support. The immobilization step, which may be performed either before or after phage binding, can also be controlled by adjusting the concentration of reactants, temperature, and time for immobilization. Capture events between biomolecules, e.g., proteins, and binding species, e.g., phage (when a phage display library is used in the DCP process), can likewise be controlled via manipulation of similar parameters.

Problems solved by technology

Unfortunately, selecting molecules of interest from large ensembles or repertoires can be time-consuming and costly, and often only provides leads which require further investigation and development.

Unfortunately, present methods of analyzing biomolecules are time-consuming and expensive, and suffer from inefficiencies in detection, imaging, purification, and analysis.

Although the genomics approach has advanced our understanding of the genetic basis of biological processes, it has significant limitations.

However, the proteomics approach faces numerous obstacles, including sample complexity, large relative abundance range, and quantification of proteins.

Technical constraints have heretofore impeded the rapid, cost-effective, reproducible, and systematic analysis of proteins and other biomolecules present in biological samples.

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